This proposal is designed to define the sex-specific negative regulation of the Drosophila male-specific lethal-1 (msl-1) gene and to characterize the possible conservation of the biochemical mechanism of Drosophila dosage compensation in mammals. The first aim is to confirm that the msl- 1 transcripts are identical in males and females using RNAse protection. The second aim is to investigate the hypothesis that the female specific Sex-lethal protein directly regulates msl-1 by binding to two of the three msl-1 transcripts, which prevents them from exiting the nucleus and/or becoming translated. To determine if there is a difference in the localization of the msl-1 transcripts in male and female cells, in situ hybridization will be performed. The potential for the transcripts to be translated will be assayed by polysome fractionation experiments. The third aim is to investigate the possible conservation of the msl-1 gene in mammals. The mammalian protein identified in cells by the Drosophila msl-1 antibody will be studied by immunofluorescence and fluorescence in situ hybridization to determine if the protein is associated with chromatin. In addition, the msl-1 mammalian homologue will be cloned and sequenced to identify potential functional domains. The results of these experiments will provide insight into general mechanisms of negative regulation as well as mammalian chromatin structure flanking actively transcribed genes.